Vibrio cholerae is a human pathogen that alternates between growth in environmental reservoirs and infection of human hosts, causing severe diarrhea. The second messenger cyclic di-GMP (c-di-GMP) mediates this transition by controlling a wide range of functions, such as biofilms, virulence, and motility. Here, we report that c-di-GMP induces expression of the extracellular protein secretion (eps) gene cluster, which encodes the type II secretion system (T2SS) in V. cholerae. Analysis of the eps genes confirmed the presence of two promoters located upstream of epsC, the first gene in the operon, one of which is induced by c-di-GMP. This induction is directly mediated by the c-di-GMP-binding transcriptional activator VpsR. Increased expression of the eps operon did not impact secretion of extracellular toxin or biofilm formation but did increase expression of the pseudopilin protein EpsG on the cell surface.IMPORTANCE Type II secretion systems (T2SSs) are the primary molecular machines by which Gram-negative bacteria secrete proteins and protein complexes that are folded and assembled in the periplasm. The substrates of T2SSs include extracellular factors, such as proteases and toxins. Here, we show that the widely conserved second messenger cyclic di-GMP (c-di-GMP) upregulates expression of the eps genes encoding the T2SS in the pathogen V. cholerae via the c-di-GMP-dependent transcription factor VpsR.KEYWORDS Vibrio cholerae, biofilm, VpsR, type II secretion, cyclic di-GMP V ibrio cholerae is a major bacterial pathogen responsible for the diarrheal disease cholera, causing an estimated 2.8 million infections each year resulting in approximately 91,000 deaths (1). V. cholerae is endemic to coastal waterways in tropical countries, where it persists in the environment as a biofilm on chitinous surfaces and periodically causes outbreaks in human populations. V. cholerae can rapidly spread and multiply under favorable environmental conditions, as seen in the recent 2010 Haiti outbreak (2). A fundamental property that allows V. cholerae to cause disease is its ability to transition from environmental reservoirs to human hosts. This transition is in part regulated by the second-messenger molecule cyclic di-GMP (c-di-GMP) (3-5).c-di-GMP is a nearly ubiquitous second-messenger molecule in bacteria that controls a range of physiological functions, including biofilm formation, motility, and virulence factor expression (6). c-di-GMP is synthesized by diguanylate cyclases (DGCs) (7) and degraded by phosphodiesterases (PDEs) (8,9). Together, these enzymes alter the concentration of c-di-GMP in the cell in response to environmental inputs. c-di-GMP has been shown to repress motility and virulence to promote a sessile, biofilmassociated lifestyle by stimulating the production of exopolysaccharide (EPS) matrix substances and adhesins while inhibiting flagellar activity or expression (3, 10-12). Levels of intracellular c-di-GMP have been proposed to be high in environmental